This project will examine gene regulation during the process of macrophage (MP) activation by the potent stimulus interferon-gamma (IFN- gamma). Over the past several years, we have been successful in cloning a number of genes that are expressed by MP in response to IFN-gamma. It is our conviction that understanding the mechanisms of regulation of such induced genes will provide an understanding of those molecular changes involved in the development of an activated MP. One model gene identified in these studies, mag-1, exhibits tissue-specific and stimulus-specific expression in response to IFN-gamma and is differentially expressed in MP at distinct differentiation stages. Expression of this gene is controlled by an unexpected pathway of IFN- gamma-mediated transcriptional regulation. Based on these initial studies, we now propose to explore three aspects of the IFN-gamma- stimulated MP activation response. We will identify the transcription factor(s) responsible for IFN-gamma-mediated induction of genes through the alternate signaling pathway, using the mag-1 promoter region as our model. We will use a variety of molecular approaches to characterize the factors binding to the critical promoter element in response to IFN-gamma and to determine the relationship of these factors to those proteins shown to be involved in the classical IFN-alpha- or IFN-gamma-mediated pathways of transcriptional regulation. We then will analyze the contribution of the mag-1 gene to the activation response induced by IFN- gamma, through the expression of this gene in normally non-expressing MP or through inhibition of gene expression. The effect of gene expression on various aspects of the MP activation response will then be monitored. We also will dissect the molecular basis of the differential regulation of gene expression during MP maturation. These studies will use both a MP cell line and primary bone marrow-derived MP as targets of chemical or cytokine induced differentiation. We then will examine the changes in IFN-gamma-mediated transcriptional regulation and induction of gene expression at different stages of MP development. The results of this work will provide novel information concerning the molecular mechanisms that control IFN-gamma induction of MP activation.